用于荧光原位杂交技术的骨髓细胞制片方法研究

摘要

This study was aimed to establish a smear protocol for preparing bone marrow cells and investigate its effect on fluorescence in situ hybridization (FISH) signal. Probe DNA (C-myc, MDM2, STK6) was labeled with Spectrum Green, PromoFluor-555 and PromoFluor-415 by nick translation. Five bone marrow samples were tested by two methods separately. Traditional method; after removing the erythrocytes by hypoosmotic solution, the bone marrow cells were fixed in methanol/acetic acid (3:1). Improved method: erythrocytes were removed using density gradient centrifugation and fixed in methanol. The samples were then fixed again in 2% formaldehyde for S min. The FISH signal was assessed by comparing the relative signal intensity of each fluorophore with the autofluorescence background. The results indicated that improved method greatly increased the ratio of fluorescence signal intensity in the Spectrum Green, PromoFluor-555 and PromoFluor-415 channel ( traditional method:4. 3 ±0. 19, 3.52 ±0.04, 3.07 ±0.08; improved method:9.89 ±0.41, 7.55 ±0.5, 5.67 ±0.18, n=5, P<0.01) respectively. The signal intensity increased 2.32,2.14 and 1.85-fold in the Spectrum Green, PromoFluor-555 and PromoFluor-415 channel respectively. In addition , the improved method decreased the split signals (traditional method:(15.8 ± 1.74)% ,(20.42 ±2. 88)% ,(23.2 ± 3.02)%;improvedmethod:(8.6±1.2)%,(12.28±1.33)%,(12.6±2.56)%, n=5, P<0.05]. It is concluded that the improved optimal procedure which facilitates FISH intensity on bone marrow cells is developed, showing potential for wide application in the diagnosis of hematologic diseases.%本研究对传统的骨髓细胞制片方法进行改良,探讨其对荧光原位杂交(FISH)信号的影响,为骨髓标本FISH检测提供最佳的制片方法.采用缺口平移的方法,制作出SpectrumGreen-C-myc,PromoFluor-555-MDM2,PromoFluor-415-STK6三色荧光杂交探针.将采集到的5例骨髓标本进行2种方法制片,传统方法:用低渗的方法去除红细胞,甲醇/冰醋酸(3:1)固定细胞后进行细胞甩片；改进方法:用密度梯度离心的方法去除红细胞,细胞甩片后用甲醇固定细胞,再用2％甲醛固定细胞,将固定好的细胞进行FISH检测并比较两种方法获得的平均荧光信号强度,背景信号强度以及荧光信号的分裂率.结果表明:在Spectrum Green,PromoFluor-555和PromoFluor-415 3个通道中,传统方法制片HSH平均荧光信号强度与背景强度的比值为4.3 ±0.19,3.52±0.04,3.07±0.08,改进方法为9.89 ±0.41,7.55±0.5,5.67±0.18,(P＜0.01)；改进方法在3个通道中获得的荧光信号强度分别为传统方法的2.32,2.14和1.85倍；同时该方法减少了荧光信号的分裂率,传统方法荧光信号的分裂率分别为(15.8±1.74)％,(20.42±2.88)％,(23.2±3.02)％,改进方法分别为(8.6±1.2)％,(12.28±1.33)％,(12.6±2.56)％(P＜0.05).结论:采用改进后的骨髓制片方法有效地提高了FISH检测的荧光信号强度,明显降低了荧光信号的分裂现象,而且该方法易于掌握,程序简单.本研究为FISH检测骨髓标本提供了一种更佳的制片方法.